Donor RBCs Research Articles

Efficacy of murine monoclonal antibodies in RBC phenotyping of DAT-positive samples

Abstract Determining the phenotype of patient RBCs that are positive by the DAT may prove problematic. Antigen typing of RBCs coated with IgG requires direct agglutinating reagents or chemical treatment (such as chloroquine diphosphate [CDP] or citric acid) to remove sufficient IgG to permit testing with IAT-reactive reagents. The citric acid elution method is commonly used in the United States; however, antigens in the Kell system are altered to the extent that they may appear to be absent by this method. There are a limited number of direct agglutinating monoclonal antibodies available. Murine monoclonal antibodies provide an additional tool for typing RBCs with a positive DAT. Five murine monoclonal IgG antibodies (anti-K: MIMA-22, MIMA-23; anti-Kpa: MIMA-21, MIMA-27; anti-Fya: MIMA-19) were used in this study. Donor RBCs with known phenotypes were sensitized in vitro with alloanti-D, alloanti-c, and alloanti-K and with 20 autoantibodies (autoanti-D [n=3], autoanti-e [n=5], autoanti-Ce/e [n=5], autoanti-e+D+E [n=1], autoanti-I [n=1], and nonspecific [n=5]) to simulate a positive in vivo DAT. The sensitized RBCs were treated with CDP to remove IgG. To determine the efficacy of the murine monoclonal antibodies when testing DAT-positive samples, both sensitized and CDP-treated RBCs were tested with these monoclonal antibodies by the IAT using anti-mouse IgG. No discrepancies were noted with the unsensitized, sensitized, or CDP-treated RBCs. An exception was noted with a potent autoanti-I, where direct agglutination of the sensitized RBCs was obtained. This study demonstrates the value of using murine monoclonal antibodies to determine the phenotype of RBCs with a positive DAT caused by autoantibodies (e.g., in autoimmune hemolytic anemia) and supports previous studies showing that RBCs sensitized in vivo can be typed without chemical manipulation. Immunohematology 2006;22:161–165.

Molecular characterization of GYPB and RH in donors in the American Rare Donor Program

Abstract Transfusion of patients with sickle cell disease (SCD) has been a challenge in clinical transfusion medicine, especially when the required donor RBCs must be U– and negative for high-prevalence Rh phenotypes (hrB, hrS). It is now possible to genotype donors to identify or confirm Uvar and U– phenotypes, as well as Rh hrB– and hrS– phenotypes, and to characterize the different RH backgrounds found in these donors. In a preliminary study of donors registered in the American Rare Donor Program, twelve different RH backgrounds were identified in eighteen hrB– or hrS– donors. These results, summarized in the current report, confirm the heterogeneous nature of these phenotypes and are relevant for selection of donor units for patients with antibodies to high-prevalence Rh antigens. Not all phenotypically similar units will be compatible, and matching the Rh genotype of the donor to the patient is important to prevent further Rh sensitization. Most donors referred were hrB– and carry at least one hybrid RHD-CE(3-7)-D gene that encodes a variant C antigen linked to RHCE*ceS that encodes the VS+V– phenotype. Surprisingly, the majority of donors were heterozygous, some even carrying conventional alleles, suggesting that the loss of expression of the hrB epitopes on RBCs is a dominant phenotype. Although antigen-matching of patients with SCD with donors for C, E, and K antigens has decreased the incidence of alloimmunization, some patients still become immunized to Rh antigens, indicating the units were not truly matched. RH genotyping can identify those patients with SCD who carry RH alleles that encode altered C, e, or D who are at risk for production of “apparent auto” and alloantibodies to Rh antigens. RH genotyping of alloimmunized patients with SCD, partnered with genotyping of donors, can identify compatible units that would also eliminate the risk of further Rh alloimmunization. Immunohematology 2006;22:143–147.

70. Long Term Correction of beta-Thalassemia by Transplantation of Transduced Hematopoietic Stem Cells

Gene therapy for beta-thalassemia requires stable transfer of the beta-globin gene into hematopoietic stem cells (HSCs) and high and regulated Hb expression in the erythroblastic progeny. We developed a novel high-titer LV to express therapeutic level of Hb in one of the most severe murine model of beta-thalassemia. We cloned the human beta globin gene under the control of a minimal promoter and 2 elements of the LCR (HS2 and HS3), in the context of a SIN-18 LV. We evaluated the efficiency of this LV to correct the thalassemic phenotype in the th3/+ mouse mutant, that exhibit chronic anemia, anomalies in red cell size and shape, splenomegaly, extramedullary hematopoiesis (EMH) and iron accumulation in liver and spleen. The beta globin transgene expression was analyzed in the differentiated progeny of transduced HSCs, after transplantation into lethally irradiated thalassemic mice. Transplantation of LV- transduced HSCs lead to a high proportion (79%|[ndash]|100%) of donor RBCs expressing human beta globin, which remained stable up to 9 months after transplantation. HPLC analysis allowed the detection of the human beta globin chain, accounting for up to 43% of endogenous murine beta globin chains. The hematocrit, red blood cell count and hemoglobin level were markedly improved (HCT 44.90|[plusmn]|6.10; RBC 9.15|[plusmn]|1.22, HGB 12.07|[plusmn]|1.75) in comparison to the hematological parameters in control mice transplanted mock- transduced th3/+ cells (HCT 29.33|[plusmn]|2.08; RBC 5.79|[plusmn]|0.45, HGB 8.03|[plusmn]|1.12). The vector derived beta-globin synthesis was able to strongly reduced abnormalities in RBC morphology in mice transplanted with LV-transduced cells. FACS analysis of the erythroid TER119+ bone marrow subpopulations showed the restoration of normal erythroblastic maturation in the gene therapy- treated mice. Finally, the histopathological analysis demonstrated the correction of splenomegaly, EMH and drastic reduction of iron deposition in the spleen and liver. Quantitative PCR analysis revealed that the average proviral copy number ranged from 0.3 to 2.2, showing a direct correlation with the degree of phenotype correction. Analysis of single erythroid CFU-S, derived from secondary recipients, confirmed that the level of vector-derived human beta-globin was copy number dependent. Moreover, Southern Blot analysis of DNA extracted from transduced bone marrow samples indicated that the engraftment of a relatively small number of transduced HSCs was sufficient to fully correct the thalassemic phenotype in the th3/+ model. Finally, we demonstrated that LV-derived human beta globin expression was persistent and rescued the thalassemic phenotype in secondary transplanted th3/+ mice. Experiments are ongoing to test the therapeutic potential of the described LV to correct thalassemia major.

In Vivo Correction of Complement Regulatory Protein Deficiency with an Inhibitor Targeting the Red Blood Cell Membrane

Because of the complement system's involvement in many human diseases and potential complications associated with its systemic blockade, site-specific regulation of this effector system is an attractive concept. We report on further developments of such an approach using a single-chain Ab fragment as a vehicle to deliver complement regulatory proteins to a defined cell type. In a model system in which RBCs deficient in complement receptor 1-related gene/protein y (Crry) are rapidly cleared after injection into wild-type animals by a complement-dependent mechanism, we selectively reconstituted these cells with N- and C-terminally targeted recombinant forms of Crry. Transfusion of Crry-coated knockout RBCs into C57BL/6 mice extended their in vivo half-life from <5 min to approximately 2 days. Maintenance of protective levels of Crry (by a combined treatment of donor and recipient RBCs) led to nearly normal RBC survival. Uniform in vitro and in vivo coating of the RBCs and the more efficient complement inhibitory capacity of C-terminally tagged Crry were other interesting features of this experimental system. These results suggest the possibility of using the single-chain Ab fragment-mediated targeting concept of complement regulatory proteins to restrict complement inhibition to the site of its excessive activation.

Nonhemolytic antibody-induced loss of erythrocyte surface antigen

AbstractTransfusion of red blood cells (RBC150) into patients with anti–donor RBC antibodies (crossmatch-incompatible transfusion) can result in lethal antibody-mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival or adversely affecting the transfusion recipient. In an effort to elucidate the mechanistic details of this process, we describe the first animal model of nonhemolytic antibody-induced RBC antigen loss. RBCs from transgenic mHEL mice express surface hen egg lysozyme (HEL) as a transmembrane protein. Transfusion of mHEL RBCs into mice immunized with HEL results in selective loss of HEL antigen from donor RBCs without affecting other blood group antigens or reducing the circulatory life span of the transfused RBCs. While this process does not require the presence of a spleen, it requires both anti-RBC immunoglobulin G (IgG) antibodies and the FcγIII receptor. These studies provide mechanistic insight into the phenomenon of antigen loss during incompatible transfusion in humans.

Anti‐D immunization by DEL red blood cells

No data are available on the immunogenicity of extremely weak D variants called DEL. Evaluation of alloanti-D formation in a D- female patient after transfusion of apparently D- blood from an Austrian donor led to discovery of a so far unknown DEL type. Standard blood group serologic methods were applied. Molecular typing, RHD sequencing, and D epitope mapping was performed and the absolute D antigen density determined. After transfusion of RBCs typed D- by routine serology, the recipient developed alloanti-D. Further evaluation with an indirect antiglobulin test confirmed donor RBCs to be D-. Molecular typing, however, demonstrated the presence of the RHD gene in one donor, and RHD sequencing revealed a deletion of four nucleotides in RHD intron 5 (RHD IVS5-38del4) as the only difference compared to the normal RHD gene. Adsorption-elution techniques demonstrated a DEL phenotype without apparent loss of D epitopes. This study documents the clinical significance of the DEL phenotype in blood units that was capable of inducing anti-D in a recipient. Qualitative data are provided on D epitope expression in DEL RBCs.

Phenotype Matching of Donor Red Blood Cell Units for Nonalloimmunized Sickle Cell Disease Patients: A Survey of 1182 North American Laboratories

The transfusion of donor red blood cell units (RBCs) that lack certain red cell antigens (such as C, E, and K) when the corresponding antigens are absent from the recipient's red cells has been shown to reduce the risk of red cell alloimmunization in sickle cell disease patients. However, data are limited regarding the extent to which transfusion services routinely perform red cell antigen phenotype testing of nonalloimmunized sickle cell disease patients, and then use that information to select donor RBCs lacking 1 or more of the red cell antigens that the patient's red cells do not express. To determine the extent to which transfusion services routinely perform red cell antigen phenotype testing of nonalloimmunized sickle cell disease patients, and then use that information to select donor RBCs lacking 1 or more of the red cell antigens that the patient's red cells do not express. An educational subsection of a College of American Pathologists Proficiency Testing Survey (J-C 2003) assessed transfusion service practices regarding performance of red cell antigen phenotype testing of nonalloimmunized sickle cell disease patients and how transfusion services use this information for the selection of donor RBCs. The data analysis of the survey included 1182 North American laboratories. Data from 1182 laboratories were included in the survey analysis, of which the majority (n = 743) reported that they did not routinely perform phenotype testing of sickle cell disease patients for antigens other than ABO and D. The other 439 laboratories reported that they did routinely perform phenotype testing of sickle cell disease patients for antigens in addition to ABO and D. The majority of these 439 laboratories (three fourths; n = 330) reported that they used these patient data for prophylactic matching with donor RBCs when sickle cell disease patients required transfusion. When phenotype-matched donor RBCs were used, the antigens most commonly matched (85% of the time) were C, E, and K. The majority of North American hospital transfusion service laboratories do not determine the red cell antigen phenotype of nonalloimmunized sickle cell disease patients beyond ABO and D. Those laboratories that do determine the red cell phenotype of nonalloimmunized sickle cell disease patients beyond ABO and D most commonly match for C, E, and K antigens when phenotype-matched donor RBCs are used.

Posttransfusion recovery of stored red blood cells in very low birth weight infants using a hemoglobin balance model.

A Hb balance model was used in very low birth weight (VLBW) infants to predict posttransfusion Hb levels from which we inferred allogeneic RBC recovery after transfusion of RBCs stored for varying periods of time. Premature VLBW infants receiving RBC transfusions during the 1st month of life were evaluated retrospectively for RBC survival of stored donor blood. Actual Hb levels measured in infant blood 1 and 2 days after RBC transfusions were compared to those predicted using a Hb balance model based on factors affecting blood Hb loss and gain. Transfusions were subgrouped according to whether or not infants were clinically stable at the time of RBC transfusion. Model-predicted RBC recovery was also evaluated relative to duration of RBC storage. Model-predicted mean (+/- SD) Hb levels 2 days after transfusion among the 30 VLBW infants receiving a total of 57 RBC transfusions were only 4 percent higher than actual values observed (15.2 +/- 1.2 g/dL vs. 14.7 +/- 1.4, respectively; p < 0.05). The infant's clinical status at the time of transfusion did not affect predicted 1- and 2-day posttransfusion RBC recovery. Model-predicted recovery of transfused RBCs was modestly, but significantly, decreased with increasing duration of donor RBC storage (i.e., 10% lower by 42 days-the maximal allowed storage period for donor blood [p < 0.01]). Our model-predicted RBC survival results are consistent with-but not direct evidence of-hemolysis of donor blood after RBC transfusion. Although observed post-RBC Hb levels 2 days after transfusion averaged only 4 percent less than predicted, model-predicted survival of donor RBCs at 42 days suggested a modest decrease (i.e., by 10%).

Deletion of arginine codon 229 in the Rhce gene alters e and f but not c antigen expression.

Rh CcEe antigens occur as ce, Ce, cE, or CE alleles in the RBC membrane. Their epitope structures and the location of their cis interacting products remain to be defined. A rare blood sample from a white male whose parents are first cousins was identified. Hemagglutination was performed using standard methods. RH structure and genotype was assessed by Southern blots. Rh transcripts were obtained by gene-specific RT-PCR and sequenced. The mutation was verified by genomic PCR assays. The donor's RBCs typed D+C-c+E-e-f(Rh6)- with a normal c dose, suggesting the Dc- phenotype. Further tests revealed a weak and qualitatively altered e expression. Southern blots indicated a genotype of Dce/dce without other gross changes. RT-PCR detected a triplet deletion (Delta685AGA687) in the Rhce gene that specifies codon 229 for arginine (Arg229). Sequencing of the region around the mutated exon 5 confirmed the donor to be homozygous for the AGA deletion. Arg229 is invariant on external loop 4 and close to the Ala226Pro change specific for e/E polymorphism. The qualitative and quantitative alteration of e antigen defines Arg229 as a crucial component for e/E epitope presentation. Given a normal dose of c antigen, the disruption of f (Rh6) by Arg229 deletion suggests that external loop 4 is a major structural element contributing to the expression of RHCE cis interacting antigenic products.

Case report: exacerbation of hemolytic anemia requiring multiple incompatible RBC transfusions

Abstract RBC transfusions in a patient with a history of autoimmune hemolytic anemia (AIHA) can represent both a laboratory and a clinical challenge. The development of high-titer low-avidity antibodies and antibodies to high-frequency antigens may further impair the ability to identify compatible donor RBCs. Not infrequently, incompatible RBCs must be used and the desire to increase oxygen carrying capacity conflicts with the desire to avoid exacerbating the autoimmune hemolytic process with RBC transfusions. A 66-year-old Caucasian female with coronary artery disease and a history of refractory AIHA had recently developed anemia and required multiple RBC transfusions. The patient had maintained adequate RBC counts with erythropoietin and prednisone therapy for the previous 16 months. With the recent worsening of her hemolytic anemia, she had developed angina that was treated with RBC transfusions in an outpatient setting. However, her angina increased as her RBC counts decreased, leading to hospital admission for further management of her hemolytic anemia and angina. She subsequently required multiple incompatbile RBC transfusions despite increased prednisone therapy and did not improve until after coronary artery stent placement and high dose IVIG therapy. This case demonstrates the usefulness of early patient phenotyping in a case of accelerating hemolytic anemia to aid in donor RBC selection, the value of communicating with clinicians and the patient regarding the use of least-incompatible RBCs, and the importance of optimizing the patient’s clinical condition to avoid ischemia. In addition, it demonstrates the value of repeated attempts with IVIG treatment despite previous refractoriness to this treatment.

Loss of enzyme-sensitive antigens due to the presence of leukocytes, neomycin sulfate, and LISS

Abstract Previous studies have shown that RBCs with residual WBCs stored in LISS and neomycin sulfate develop characteristics associated with enzyme-treated RBCs. During a mass screening program to antigen type donor RBCs, we observed that the Fya antigens on a RBC sample from an in-house panel became non-detectable with anti-Fya after incubation overnight in Diluent 2 from Micro Typing Systems, Inc. (MTS, Pompano Beach, FL). In response to this observation, we initiated an investigation to determine the cause. Tests were performed according to the manfacturer’s instructions in MTS neutral gel cards or gel cards containing anti-IgG. We found that a reduction or loss of the Fya, Fyb, and M antigens occurs when RBCs were prepared from samples containing residual WBCs (as a source of enzymes) and subsequently incubated in media containing neomycin sulfate and LISS. We showed that the effect did not occur in the absence of neomycin sulfate. RBC antigens can be altered in LISS if they have first been exposed to neomycin. We recommend restricting the use of RBCs suspended in MTS Diluent 2 to the day of dilution (as indicated in the package insert) if preparing reagent RBCs from sources that were not leukoreduced and were stored in the presence of neomycin.

Sickle Hb polymerization in RBC components from donors with sickle cell trait prevents effective WBC reduction by filtration.

RBC components collected from donors with sickle cell trait frequently occlude WBC-reduction filters. In vitro, sickle trait RBCs have the potential for sickle Hb (Hb S) polymerization at low oxygen saturations and high Hb concentrations. To determine if the low pH and high osmolarity of the CP2D used in the collection contributed to filter failures, the filterability of sickle trait donor RBCs collected in CP2D was compared with RBCs from the same donors collected in heparin. Five of six sickle trait components collected in CP2D did not complete filtration, but all six RBC components collected in heparin filtered completely. RBC components collected in CP2D from four other sickle trait donors were divided in two, and one-half was treated with carbon monoxide to convert Hb S to its liganded form to prevent Hb S polymerization. All four carbon monoxide-treated components filtered within 9 minutes, but only one untreated component filtered completely. RBC components collected by apheresis contained less CP2D, and five of seven sickle trait apheresis components filtered completely; four of the five filtered rapidly (<15 min) and one filtered in 100 minutes. Hb oxygen saturation was greater in the four rapidly filtering apheresis RBC components (68 +/- 9%) than in the three that filtered slowly or incompletely (37 +/- 5%, p = 0.03). Hb S polymerization appears responsible for RBC WBC-reduction filter failures. Citrate anticoagulant and low oxygen saturation are responsible in part for Hb S polymerization in this setting.

K, Fy(a), and Jk(a) phenotyping of donor RBCs on microplates.

In many cases, the search for compatible blood for patients with clinically significant RBC alloantibodies is difficult and time-consuming. To date, it has been considered necessary only to phenotype the blood donors for ABO group and D. There has been long experience with automated routine analysis (ABO, C, c, D, E, and e typing and RBC antibody screening), using robotic dispensers and computerized interpretation of microplate results. The purpose of this study was to explore the possibilities of also phenotyping for K, Fy(a), and Jk(a), as antibodies directed against these antigens (together with Rh antigens) are the most common clinically significant alloantibodies in the Swedish population. One thousand thirty-one EDTA samples from blood donors were phenotyped for K, Fy(a), and Jk(a) by use of an IAT with PEG on microplates. The findings were compared to those using conventional IAT in tube's and the microcolumn gel test (DiaMed-ID, DiaMed). All typing results with the microplate method were correct. All reactions for K and Fy(a) typing could be interpreted by the computer. The results for Jk(a) were indeterminate in 1.4 percent (14/1031) of the samples. The PEG-IAT microplate method gave reliable results that were suitable for routine phenotyping, thus making available a stock of phenotyped blood at reasonable cost, ready for delivery when required.

Apparent rejuvenation of transfused donor blood in the fetus is due to accelerated removal of the older RBCs.

In severely anemic fetuses of women alloimmunized to RBC antigens, transfused donor RBCs disappear faster than in adults. This may result from an accelerated linear or nonlinear decline with time. It was investigated whether changes in donor RBC age characteristics after circulation in the fetus may reflect the main type of cellular decline. Donor RBC age characteristics (density, mean cell volume [MCV], and mean cell Hb content [MCHC]) were determined before intrauterine transfusions. Density gradient centrifugation was used to obtain RBCs of different ages. The results from gradient centrifugation were used to calculate mean values for the density, MCV, and MCHC to be expected after the transfusion interval, assuming a linear decline in RBCs of 1 percent per day. Donor and fetal RBCs, taken just before the second transfusion, were separated by agglutination with IgM D MoAb. For these donor cells, the observed mean values for density, MCV, and MCHC were compared with the calculated, expected values (n = 12). The mean +/- SD transfusion interval was 17.9 +/- 3.6 days. The Hb declined by 1.75 +/- 0.62 percent per day (n = 9). After the transfusion interval and contrary to the expected changes, cell density and MCHC decreased and MCV increased significantly (0. 001<p<0.02). This difference between actual and calculated values decreased with increasing intervals; for MCV, it was also associated with a greater decline in Hb per day (p<0.05). All donor cells age during circulation in the fetus. However, after the transfusion interval, the donor RBC population remaining is apparently younger than the RBC population before transfusion. This results from a preferential disappearance of older donor RBCs and not from a linear loss of cells with time. The removal of older RBCs before the transfusion may increase the time between transfusions and thereby reduce the total number of transfusions required.

The effect of the source of transfused blood on rate of consumption of transfused RBC's in pregnancies affected by RBC isoimmunization

37 THE EFFECT OF THE SOURCE OF TRANSFUSED BLOOD ON RATE OF CONSUMPTION OF TRANSFUSED RBC'S IN PREGNANCIES AFFECTED BY RBC ISOIMMUNIZATION. S. ElAzeem, R.L Rose, P. Samuds and R.D~ O'Shaughnessy The Ohio State Universirf Hospitals, Colnmbus, Ohio OBJECTIVE: To determine if there is an advantage to using maternal RBCs instead of donor RBCs in treating isoinmrunized fetuses. STUDY DESIGN: The daily consmnption of hemoglobin was calculated in 292 intravascular fetal transfusions performed in 53 pregnancies in 44 patients over 9 years. The rates of consmnption were compared using unpaired t-tests. The results were stratified according to the gestational age groups 18-24 weeks, 25-28 weeks, 29-32 weeks and 33-38 weeks. RESULTS: The accompanying table shows the number of observations in each group.

Induction of additional red cell alloantibodies after intrauterine transfusions.

The aim of this study was to determine the frequency and origin of additional alloantibodies directed against red cells (RBCs) after intrauterine transfusion (IUT). Between March 1987 and December 1992, fetuses with severe hemolytic disease (n = 91) received a total of 280 ultrasound-guided IUTs of RBCs from unrelated donors. The specificity of alloantibodies to RBCs in maternal serum was determined both before and after each IUT. If additional alloantibodies directed against RBCs were detected, their origin was determined by phenotyping the fetal, donor, and paternal RBCs for each particular antigen. The study included a control group of 69 pregnant women who underwent either amniocentesis or fetal blood sampling. Production of additional alloantibodies directed against RBC antigens was detected in 24 women (26%). The source of the immunizing antigen, either donor or fetus, was identified in 14 patients. The additional alloantibodies were directed against fetal antigens in 11 women and against donor antigens in 3. One additional alloantibody directed against donor antigen clearly reduced the survival of donor RBCs. The fetus and the donor shared the immunizing antigen in four cases, and in one case, the antibody occurred naturally. In five cases, the source of the immunizing antigen was not determined. In the control group, additional antibodies were detected in two patients. IUT therapy is associated with a high incidence of additional alloantibodies. In the majority of patients, the use of maternal RBCs for IUT would not have prevented additional formation of alloantibody to RBCs.

Effect of delayed centrifugation or reading on the detection of ABO incompatibility by the immediate‐spin crossmatch

The immediate-spin (IS) crossmatch is a method that detects ABO incompatibility. However, under certain circumstances, this test may show unwanted negative or weak results, even though the red cells (RBCs) and serum being tested are ABO incompatible with each other. The present study investigated the potential effect of delayed centrifugation or reading on the IS crossmatch test performance. When the centrifugation step of the IS crossmatch between group O sera and group A1 RBCs was delayed for 2 minutes, 5 of 200 crossmatches showed no agglutination with only trace (n = 2) or moderate (n = 3) hemolysis, and one crossmatch showed only weak, macroscopic agglutination, but moderate hemolysis. All six sera contained A antibodies that were lytic in vitro and, therefore, probably were capable of causing in vivo hemolysis of transfused A1 RBCs. Delaying the reading of the IS crossmatch test for 2 minutes had no apparent effect on test performance. These data demonstrate the importance of technologists' recognition of hemolysis as a positive result on IS crossmatches, especially if the performance of the centrifugation step of the test is delayed. Furthermore, the unwanted negative agglutination results were abolished by suspending the group A1 RBCs in saline containing EDTA. The authors' laboratory has modified its IS crossmatch procedure so that donor RBCs are routinely suspended in saline containing EDTA before testing. This procedural change should increase the safety of the IS crossmatch.

Presence of P blood group antigens on human platelets.

Except for ABH antigens, the presence of red cell (RBC) antigens on human platelets has been a source of disagreement among investigators. Because ABH antigens share precursor sequences with P-system saccharides, the authors examined human platelets for evidence of P blood group antigens. Anti-P was directly labeled with 125I and incubated with normal platelets in a one-stage radioimmunoassay (RIA). Alternatively, platelets from donors of known RBC phenotype were incubated with anti-P, anti-P1, or anti-Tja, washed, incubated with FITC-labeled goat anti-human immunoglobulin and evaluated by fluorescence flow cytometry. The results of these assays demonstrate that platelets express P blood group antigens in parallel to the donor's RBCs. The role of these antigens in platelet transfusion is not known.

Combined Sickle Cell Disease and Autoimmune Hemolytic Anemia

Four patients experienced combined sickle cell disease and autoimmune hemolytic anemia within the past ten years. A fifth patient had positive direct antiglobulin test results without verified autoimmune hemolysis. Severely accelerated hemolysis was observed in four patients; anemia was severe, and the reticulocyte count rose into the 60% to 88% range. During the period of active autoimmune hemolysis, decline of the reticulocyte count rose into the 60% to 88% range. During the period of active autoimmune hemolysis, decline of the reticulocyte count into the 6% to 16% range was associated with rapid decrease in the hemoglobin level, requiring transfusion. All five patients were already alloimmunized by transfusions administered before onset of the autoimmune hemolytic anemia; two or more allospecificities were identified in four of five patients. The presence of autoantibody notably compromised compatibility testing; three patients experienced posttransfusion hemoglobinuria, and in vivo cross matching with 51Cr-labeled donor RBCs was employed on three occasions. All patients responded to corticosteroids; mercaptopurine was also administered to one patient. The direct antiglobulin test result reverted to negative in all patients after hospital discharge.

Prevention of Rh Immunization

SummaryFour-hundred-and-fifty µgm of Rh0(D) antibody given I.M. 24 hours before transfusion of 2 ml of Rh-positive into Rh-negative ABO compatible recipient, causes rapid removal of the transfused cells. Fifty percent of the injected RBCs disappeared from the circulation with a mean T-V2 of 8 minutes (range 4-12 min). N o circulating donor RBCs were detected in the peripheral blood 4 hours after transfusion. The destruction of trapped 51Cr-labelled cells occurs over a three-day period primarily in the liver and spleen, presumably in the reticuloendothelial cells. Once trapped in the reticuloendothelial system, intact cells do not re-enter the circulation. Chromium-51 liberated after destruction of RBCs enters the plasma and is rapidly eliminated in the urine. No untoward reaction either local or systemic occurred after intramuscular injection of Rh0(D) antibody. Clinical trials to determine the minimum effective dose of antibody for routine and "high risk" group are needed but it is likely to be well below the dose of 450 µgm used in this study.